Plant regeneration from cultured cells of the great majority of monocot (mostly graminaceous) species that have been reported so far, is achieved from callus initiated on high concentrations of a strong auxin, such as 2,4-dichlorophenoxyacetic acid (2,4-D) (Conger, B. V. et al., Current Issues in Plant Molecular and Cellular Biology, pp. 59-68 (1995)). The synthetic auxin, 2,4-D, is considered to be the best plant hormone to induce embryogenic callus. Embryogenic callus is typically obtained from monocots by inducing the primary cell culture on a medium containing one or more auxin-type plant hormones followed by a secondary cultivation step on a lowered auxin but in the presence of a cytokinin-type plant hormone. The embryogenic potential of monocot cell cultures diminishes over time, making it necessary to reinitiate the primary cell culture (U.S. Pat. No. 6,153,812 issued Nov. 28, 2000; Trigiano and Gray, Plant tissue culture concepts and laboratory exercises. Second ed., Boca Raton: CRC Press (2000)).
Plants from the Class Monocotyledoneae are often multipurpose plants. Giant reed, Arundo donax, for example, has been used for 5,000 years for pipe instruments and is the source for reeds for clarinets and organ pipes. Even with today's modern technology, most of the reeds for woodwind musical instruments are still made from A. donax culms.
Giant reed is also used for erosion control and has great potential for use as an energy crop (Szabo, P., et al., J. Anal. Appl. Pyrolysis, 36:179-190 (1996)). The culms are also used for fishing rods, walking sticks, mats and lattices in the construction of adobe huts. Giant reed is also a source of industrial cellulose for paper and rayon making, and for the production of other polysaccharides (Neto, C. P. et al., Ind. Crops & Prods., 6:51-58 (1997). It has even been considered as a source of pulp for the making of paper. Additionally, giant reed can also be used in bioremediation efforts to remove environmental pollutants from water and land areas.
Giant reed is only one of many monocots that exhibit such multiple uses. Whether used as ornamentals, sources of energy, or as useful vehicles to carry out industrial processes, such grass-like plants are important.
The present inventors have found that 2,4-D induced callus cultures obtained from giant reed nodal segments and segments of immature inflorescences fail to produce sustained embryogenic cell cultures and embryos even upon transfer to low auxin or no auxin in combination with a cytokinin, as is the typical practice (U.S. Pat. No. 6,153,812 issued Nov. 28, 2000; Trigiano and Gray, Plant tissue culture concepts and laboratory exercises. Second ed., Boca Raton: CRC Press (2000)). The finding that these methods fail to produce embryogenic cultures is corroborated by Tóth and Mix-Wagner and Linder and Gallagher where Tóth and Mix-Wagner reported the formation of callus that was claimed to be embryogenic but they failed to obtain embryos and plant regeneration and Linder and Gallagher announced one-time plant regeneration from callus without the potential for mass propagation (Tóth and Mix-Wagner, Sustainable agriculture for food, energy and industry: strategies towards achievement: proceedings of the international conference held in Braunschweig, Germany, June 1997, N. El Bassarn, et al., Eds., James & James (Science Publishers) Ltd.: London. pp. 249-253 (1998); Linder and Gallagher, Abstract No. 257, American Journal of Botany, 85(6): 89 (1998)).
Accordingly, it would be useful to have methods for the large-scale micropropagation and macropropagation of Arundo donax and other monocot plants. Thus, the present invention provides a comprehensive set of methods for the production, propagation, maintenance, storage, transportation and deployment of sustained embryogenic cell cultures as well as derived secondary and tertiary totipotent tissue cultures from plant species of the Class Monocotyledoneae and from elite plant lines derived therefrom. The plant micropropagation technology of the present invention based on the sustained embryogenic cultures provides unprecedented efficiency.